The tethered agonist approach to mapping ion channel proteins – toward a structural model for the agonist binding site of the nicotinic acetylcholine receptor.
Research by Lintong Li, Wenge Zhong, Niki Zacharias, Caroline Gibbs, Henry A Lester, Dennis A. Dougherty.
Condensation of the Research
Purpose of the Study:
The purpose of the study was to develop a structural model for the nicotinic acetylcholine receptor and use newer techniques to more easily understand the structure and function of membrane proteins.
Understanding the integral membrane proteins of neurons and excitable cells is often difficult because they cannot be studied using tradition techniques of X-ray crystallography and NMR. Therefore to understand their function other techniques must be used to study them. The most useful approach that has been determined to analyze these proteins is to use two, site-directed mutagenesis and electrophysiology. This approach has been improved by nonsense suppression methodology. With this a tethered agonist approach was used to map the agonist binding site of ligand-gated channels. This would provide invaluable information for understanding its response to pharmaceuticals and other chemicals in the human system. Previous studies indicated that sub-units near the binding site of the nicotinic acetylcholine receptor may contribute to binding and may actually be the binding sites. Using the new method of tethering unnatural amino acids can provide information in determining where and how the binding site works. The use of unnatural amino acids allows researchers to incorporate them in multiple positions to pinpoint tether locations and eliminate proposed sites. With new studies a better model for the agonist binding site of the nicotinic acetylcholine receptor can be developed. Proteins with synthesized, unnatural amino acids were expressed on Xenopus oocytes to study the structure and function of the binding site.
1. Synthesis of Tyr-OnQ and Tyr-O3Bu
In the unnatural amino acid technique the target is to the develop an “amino acid with the side chain in place, the amino group protected, and the carboxylate activated as a cyanomethyl ester. The usual method of forming the cyanomethyl ester in the final step failed. It was found that the quaternary ammonium had to be added to the final step of the synthesis. The synthesis of Tyr-O2Bu was not complicated with other obstacles.
2. Incorporationof Tyr-OnQ into the nAChR
The unnatural amino acid Tyr-OnQ was added at multiple position of the receptor site. For sites designated as á two copies of the synthesized side chain was used. For ã and ä sites mutations were made to attach there. This was done so that both agonist binding sites were disturbed. With there unnatural amino acids mutant proteins were expressed on the Xenopus oocytes. Once expressed their whole...